The low calcium response (LCR) is an essential determinant of virulence in Yersinia pestis, the causative agent of plaque, and in two other human pathogens of the same genus, Y. entercolitica and Y. psuedotuberculosis. LCR is observed as cessation of growth and expression of two novel proteins, the V and W antigens, when cultures in Ca2+ free medium are shifted from 26 degrees C to 37 degrees C. Expression of a group of outer membrane proteins is also associated with the LCR genes. LCR is thought to be triggered by the low free Ca2+ concentrations encountered when Y. pestis enters the intracellular environment and thus, rather than restricting growth, LCR may aid the bacteria in adaptation to intracellular conditions. We have recently observed a cytotoxic effect in Y. pestis specific to the plasmid which encodes LCR. This effect could be the major contribution of LCR to virulence. This project will focus on the cytotoxic effect, V antigen, and regulation of LCR by temperature and by Ca2+ concentration. The genes involved in each of these phenomena will be identified, their function determined and their importance for expression of virulence assessed. These goals will be accomplished by detailed genetic analysis of mutants with specific defects in expression of cytotoxicity or in regulation of LCR, coupled with virulence testing in mice as well as in vitro analysis of phenotypes. For example, regulation by Ca2+ will be investigated with mutants defective in their ability to detect Ca2+. Such mutants still exhibit temperature regulation and express the other features of LCR at 37 degrees C whether or not Ca2+ is present. Additionally, operon fusion strains constructed for analysis of Ca2+ regulation will be used to determine whether or not Ca2+ regulated genes are induced by entry of Y. pestis into animal cells. It is becoming increasingly clear that regulation of virulence genes is an important but poorly studied aspect of pathogenesis. This project will provide one of the first detailed descriptions of the regulation employed by a facultative intracellular pathogen. It may also establish cytotoxicity as the major contribution of LCR to virulence.